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( A ) Based on data from the DepMap portal, the averag e expression of the YPEL gene family members relative to the expression of YPEL1 in breast cancer cell lines (DM-BCCL) or only in MCF7 cells (DM-MCF7) is shown. For comparison, our RT-qPCR analyses for the relative expression of YPEL genes in MCF7 cells (MCF7) are also indicated. ( B ) To assess whether or not E2-ERα signaling is involved in the regulation of YPEL2 , 3 , and 5 expressions, MCF7 cells grown for 72 h in CD-FBS containing growth medium were treated without (EtOH as control) or with 10 -9 M E2, and/or 10 -9 M ICI, a complete ER antagonist, for 24h. cDNAs generated from total RNA were subjected to qPCR. We also used the E2-ERα responsive gene TFF1 as a control. Star indicates a significant change compared to EtOH control. ( C ) Relative to YPEL1 , the expression of YPEL genes in COS7 cells was assessed by qPCR using cDNA from total RNA obtained from COS7 cells grown in steady-state conditions. ( D ) To evaluate the synthesis of YPEL1-5 proteins, which Y3.1 and Y3.2 indicate short and long variant YPEL3 proteins, COS7 cells were transiently transfected with the expression vector pcDNA3.1(-) bearing none (EV) or cDNA for a YPEL protein. Total protein extracts of COS7 cells grown in steady-state condition were subjected to SDS-15%PAGE followed by WB using a pan-YPEL antibody (SCBT, sc99727) and an HRP-conjugated goat-anti-rabbit secondary antibody (Advansta R-05072–500). Membranes were re-probed with an antibody specific to <t>HDAC1</t> (Abcam, <t>ab19845).</t> Molecular masses (MM) in kDa are indicated. ( E ) To assess the synthesis of endogenous YPEL proteins in COS7 cells, protein extracts ranging from 50 to 150 µg were subjected to WB using the pan-YPEL antibody. We also used protein extracts from COS7 cells transiently transfected with pcDNA3.1(-) bearing the 3F-YPEL2 cDNA for comparison. Membranes were re-probed with the HDAC1 antibody (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( F ) To assess the intracellular localization of endogenous YPEL (YPEL) proteins, COS7 cells grown on coverslips were subjected to ICC using the pan-YPEL antibody followed by an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Abcam, ab150080) or a vimentin (sc-6260) antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113). DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( G & H ) The intracellular localization of 3F-YPEL2 was evaluated in transiently transfected cells with the use of the Flag ( G ) or the pan-YPEL ( H ) antibody or GFP fusion ( I ). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Rabbit Polyclonal Hdac1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance"

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

Journal: bioRxiv

doi: 10.1101/2023.07.31.551286

Figure Legend Snippet: ( A ) Based on data from the DepMap portal, the averag e expression of the YPEL gene family members relative to the expression of YPEL1 in breast cancer cell lines (DM-BCCL) or only in MCF7 cells (DM-MCF7) is shown. For comparison, our RT-qPCR analyses for the relative expression of YPEL genes in MCF7 cells (MCF7) are also indicated. ( B ) To assess whether or not E2-ERα signaling is involved in the regulation of YPEL2 , 3 , and 5 expressions, MCF7 cells grown for 72 h in CD-FBS containing growth medium were treated without (EtOH as control) or with 10 -9 M E2, and/or 10 -9 M ICI, a complete ER antagonist, for 24h. cDNAs generated from total RNA were subjected to qPCR. We also used the E2-ERα responsive gene TFF1 as a control. Star indicates a significant change compared to EtOH control. ( C ) Relative to YPEL1 , the expression of YPEL genes in COS7 cells was assessed by qPCR using cDNA from total RNA obtained from COS7 cells grown in steady-state conditions. ( D ) To evaluate the synthesis of YPEL1-5 proteins, which Y3.1 and Y3.2 indicate short and long variant YPEL3 proteins, COS7 cells were transiently transfected with the expression vector pcDNA3.1(-) bearing none (EV) or cDNA for a YPEL protein. Total protein extracts of COS7 cells grown in steady-state condition were subjected to SDS-15%PAGE followed by WB using a pan-YPEL antibody (SCBT, sc99727) and an HRP-conjugated goat-anti-rabbit secondary antibody (Advansta R-05072–500). Membranes were re-probed with an antibody specific to HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( E ) To assess the synthesis of endogenous YPEL proteins in COS7 cells, protein extracts ranging from 50 to 150 µg were subjected to WB using the pan-YPEL antibody. We also used protein extracts from COS7 cells transiently transfected with pcDNA3.1(-) bearing the 3F-YPEL2 cDNA for comparison. Membranes were re-probed with the HDAC1 antibody (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( F ) To assess the intracellular localization of endogenous YPEL (YPEL) proteins, COS7 cells grown on coverslips were subjected to ICC using the pan-YPEL antibody followed by an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Abcam, ab150080) or a vimentin (sc-6260) antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113). DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( G & H ) The intracellular localization of 3F-YPEL2 was evaluated in transiently transfected cells with the use of the Flag ( G ) or the pan-YPEL ( H ) antibody or GFP fusion ( I ). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Techniques Used: Expressing, Comparison, Quantitative RT-PCR, Control, Generated, Variant Assay, Transfection, Plasmid Preparation, Staining

$( A ) COS7 cells were transiently transfected with the pINDUCER20-MCS vector bearing none (EV) or the 3F-YPEL2 cDNA for 24 h. Cells were then treated without (0) or with varying concentrations (1-1000 ng/ml) of Dox for 24 h. Total cell extracts (50 µg) were then subjected to WB using the Flag antibody followed by an HRP-conjugated goat-anti-mouse secondary antibody (Advansta R-05071-500). Membranes were re-probed with the HDAC1 antibody. Molecular masses (MM) in kDa are indicated. ( B ) Transiently transfected cells with pINDUCER20-MCS bearing the 3F-YPEL2 cDNA for 24 h were then treated with 0, 5, 10, or 100 ng/ml Dox for 24 h. Cells were subsequently fixed, permeabilized, washed, and stained with the Flag antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113) or a Lamin B1 antibody (Abcam, ab16048) followed by an Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( C ) To assess the subcellular levels of inducibly synthesized 3F-YPEL2 in transiently transfected cells treated without or with various concentrations of Dox for 24 h, fractionated nuclear and cytoplasmic protein extracts were subjected to WB using the Flag antibody. Membranes were re-probed with an antibody specific to β-actin (Abcam, ab8227) or HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( D ) To assess the effects of 3F-YPEL2 on cellular growth, COS7 cells transiently transfected with pINDUCER20-MCS bearing none (EV) or the 3F-YPEL2 cDNA for 24 h were treated without or with 10 ng/ml Dox for 24 h intervals up to 96 h. At every 24 h, cells were collected and counted with a hematocytometer. The asterisk indicates significant changes (P<0.05) in cellular growth depicted as fold changes. ( E ) Crystal violet staining for cellular growth and ( F ) WB analysis with the Flag antibody of COS7 cells inducibly synthesizing 3F-YPEL2 at 96 h are shown.$
Figure Legend Snippet: ( A ) COS7 cells were transiently transfected with the pINDUCER20-MCS vector bearing none (EV) or the 3F-YPEL2 cDNA for 24 h. Cells were then treated without (0) or with varying concentrations (1-1000 ng/ml) of Dox for 24 h. Total cell extracts (50 µg) were then subjected to WB using the Flag antibody followed by an HRP-conjugated goat-anti-mouse secondary antibody (Advansta R-05071-500). Membranes were re-probed with the HDAC1 antibody. Molecular masses (MM) in kDa are indicated. ( B ) Transiently transfected cells with pINDUCER20-MCS bearing the 3F-YPEL2 cDNA for 24 h were then treated with 0, 5, 10, or 100 ng/ml Dox for 24 h. Cells were subsequently fixed, permeabilized, washed, and stained with the Flag antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113) or a Lamin B1 antibody (Abcam, ab16048) followed by an Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( C ) To assess the subcellular levels of inducibly synthesized 3F-YPEL2 in transiently transfected cells treated without or with various concentrations of Dox for 24 h, fractionated nuclear and cytoplasmic protein extracts were subjected to WB using the Flag antibody. Membranes were re-probed with an antibody specific to β-actin (Abcam, ab8227) or HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( D ) To assess the effects of 3F-YPEL2 on cellular growth, COS7 cells transiently transfected with pINDUCER20-MCS bearing none (EV) or the 3F-YPEL2 cDNA for 24 h were treated without or with 10 ng/ml Dox for 24 h intervals up to 96 h. At every 24 h, cells were collected and counted with a hematocytometer. The asterisk indicates significant changes (P<0.05) in cellular growth depicted as fold changes. ( E ) Crystal violet staining for cellular growth and ( F ) WB analysis with the Flag antibody of COS7 cells inducibly synthesizing 3F-YPEL2 at 96 h are shown.

Techniques Used: Transfection, Plasmid Preparation, Staining, Synthesized

Figure Legend Snippet: ( A ) COS7 cells were transiently transfected with the pINDUCER20-MCS vector bearing none (EV) or the 3F-YPEL2 cDNA for 24 h. Cells were then treated without or with 10 ng/ml of Dox every 24 h for 96 h. Total cell extracts (50 µg) were then subjected to WB using the Flag antibody. Membranes were re-probed with the HDAC1 antibody. Molecular masses (MM) in kDa are indicated. ( B ) Transfected cells treated without or with 10 ng/ml of Dox for 24 h were also subjected to cell cycle analysis and ( C ) Annexin V staining. ( D ) Un-transfected cells were also treated with 10 or 100 µM apoptosis inducer Doxorubicin for 24h as control.

Techniques Used: Transfection, Plasmid Preparation, Cell Cycle Assay, Staining, Control

( A ) COS7 cells were transfected with pINDUCER20-MCS carrying none (EV), the 3F-YPEL2 (3F-Y2), 3F-YPEL2-Turbo-HA (3F-Y2-T-HA), or Turbo-HA (T-HA) cDNA. 24 hours after transfection, cells were treated without or with 10 ng/ml Dox to induce protein synthesis for 24 h in the absence or presence of biotin (50 μM) for the biotinylation of endogenous proteins. Cells were then collected and equal amounts (50 µg) of protein extracts were subjected to SDS-10%PAGE electrophoresis followed by WB analyses using the Flag, HA, or Biotin (Abcam, ab53494) antibody. Molecular masses are indicated in kDa. ( B ) To assess subcellular distributions of 3F-YPEL2 (3F-Y2), Turbo-HA (T-HA), or 3F-YPEL2-Turbo-HA (3F-Y2-T-HA) proteins following 10 ng/ml Dox induction for 24 h in transiently transfected cells, cytoplasmic, nuclear, or the total protein extracts (50 µg) were subjected to WB analyses wherein β-actin (β-Ac, ab8227) and HDAC1 (HD1) were used as the cytoplasmic and nuclear protein loading controls. Molecular masses are depicted in kDa. ( C ) Intracellular locations of the TurboID constructs in the transiently transfected cell grown on coverslips without (-) with Biotin (+) were assessed with the Flag antibody followed by the Alexa Fluor 488-conjugated secondary antibody (Abcam, ab150113), or HA and/or biotin antibody followed by the Alexa Fluor 594-conjugated secondary antibody (Abcam, ab150080). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Figure Legend Snippet: ( A ) COS7 cells were transfected with pINDUCER20-MCS carrying none (EV), the 3F-YPEL2 (3F-Y2), 3F-YPEL2-Turbo-HA (3F-Y2-T-HA), or Turbo-HA (T-HA) cDNA. 24 hours after transfection, cells were treated without or with 10 ng/ml Dox to induce protein synthesis for 24 h in the absence or presence of biotin (50 μM) for the biotinylation of endogenous proteins. Cells were then collected and equal amounts (50 µg) of protein extracts were subjected to SDS-10%PAGE electrophoresis followed by WB analyses using the Flag, HA, or Biotin (Abcam, ab53494) antibody. Molecular masses are indicated in kDa. ( B ) To assess subcellular distributions of 3F-YPEL2 (3F-Y2), Turbo-HA (T-HA), or 3F-YPEL2-Turbo-HA (3F-Y2-T-HA) proteins following 10 ng/ml Dox induction for 24 h in transiently transfected cells, cytoplasmic, nuclear, or the total protein extracts (50 µg) were subjected to WB analyses wherein β-actin (β-Ac, ab8227) and HDAC1 (HD1) were used as the cytoplasmic and nuclear protein loading controls. Molecular masses are depicted in kDa. ( C ) Intracellular locations of the TurboID constructs in the transiently transfected cell grown on coverslips without (-) with Biotin (+) were assessed with the Flag antibody followed by the Alexa Fluor 488-conjugated secondary antibody (Abcam, ab150113), or HA and/or biotin antibody followed by the Alexa Fluor 594-conjugated secondary antibody (Abcam, ab150080). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Techniques Used: Transfection, Electrophoresis, Construct, Staining

( A-C ) To assess the interactions of 3F-YPEL2 with putative interaction partners, the expression vector pcDNA3.1(-) bearing the 3F-YPEL2, ( A ) HA-ADSS, ( B ) HA-EEF1D, or ( C ) HA-G3BP1 cDNA were transiently transfected into COS7 cells for 24h. The synthesis of proteins was assessed by WB using the Flag (F-1804) or the HA (ab9119) antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody (ab19845). Cellular extracts (500 μg) of transiently co-transfected cells were subjected to Co-IP with the Flag, HA, or isotype-matched IgG. The precipitates were then subjected to SDS-10%PAGE followed by WB using the HA or Flag antibody. Molecular masses (MM) in kDa are indicated.

Figure Legend Snippet: ( A-C ) To assess the interactions of 3F-YPEL2 with putative interaction partners, the expression vector pcDNA3.1(-) bearing the 3F-YPEL2, ( A ) HA-ADSS, ( B ) HA-EEF1D, or ( C ) HA-G3BP1 cDNA were transiently transfected into COS7 cells for 24h. The synthesis of proteins was assessed by WB using the Flag (F-1804) or the HA (ab9119) antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody (ab19845). Cellular extracts (500 μg) of transiently co-transfected cells were subjected to Co-IP with the Flag, HA, or isotype-matched IgG. The precipitates were then subjected to SDS-10%PAGE followed by WB using the HA or Flag antibody. Molecular masses (MM) in kDa are indicated.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Control, Co-Immunoprecipitation Assay

( A ) To assess the intracellular localization of 3F-YPEL2 and/or HA-ELAVL1, COS7 cells grown on coverslips un-transfected (UT) or transiently transfected for 36 h with the expression vector pcDNA3.1(-) bearing the 3F-YPEL2 or HA-ELAVL1 cDNA were stained with the Flag or the HA antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( B ) To examine the protein synthesis, COS7 cells were transfected (+) with the expression vector bearing 3F-YPEL2 and/or HA-ELAVL1 cDNA. The synthesis of proteins was assessed by WB using the Flag or the HA antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody. ( C ). The cellular extracts (500 μg) of transiently co-transfected COS7 cells were subjected to Co-IP with the HA, Flag, or isotype-matched IgG. 50 μg of lysates was used as input control. The precipitates were subjected to SDS-10%PAGE followed by WB using the Flag or the HA antibody. Molecular masses (MM) in kDa are indicated. ( D ) To assess in cellula interaction of 3F-YPEL2 and HA-ELAVL1, the proximity ligation assay was carried out in transiently transfected COS7 cells grown on coverslips. Cells were fixed, permeabilized, blocked, and probed with the HA and/or the Flag antibody. Cells were then subjected to fluorescent probes for circular DNA amplification for proximity interaction foci. DAPI was used for nuclear staining. Images were captured with a fluorescence microscope. The scale bar is 20 µm. ( E ) The intracellular localization, ( F ) protein synthesis, ( G ) interaction, and ( H ) PLA of 3F-YPEL2 and/or HA-SQSTM1 in transiently transfected COS7 cells were as assessed as described in legend of A-D. HDAC1 was used as a loading control.

Figure Legend Snippet: ( A ) To assess the intracellular localization of 3F-YPEL2 and/or HA-ELAVL1, COS7 cells grown on coverslips un-transfected (UT) or transiently transfected for 36 h with the expression vector pcDNA3.1(-) bearing the 3F-YPEL2 or HA-ELAVL1 cDNA were stained with the Flag or the HA antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( B ) To examine the protein synthesis, COS7 cells were transfected (+) with the expression vector bearing 3F-YPEL2 and/or HA-ELAVL1 cDNA. The synthesis of proteins was assessed by WB using the Flag or the HA antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody. ( C ). The cellular extracts (500 μg) of transiently co-transfected COS7 cells were subjected to Co-IP with the HA, Flag, or isotype-matched IgG. 50 μg of lysates was used as input control. The precipitates were subjected to SDS-10%PAGE followed by WB using the Flag or the HA antibody. Molecular masses (MM) in kDa are indicated. ( D ) To assess in cellula interaction of 3F-YPEL2 and HA-ELAVL1, the proximity ligation assay was carried out in transiently transfected COS7 cells grown on coverslips. Cells were fixed, permeabilized, blocked, and probed with the HA and/or the Flag antibody. Cells were then subjected to fluorescent probes for circular DNA amplification for proximity interaction foci. DAPI was used for nuclear staining. Images were captured with a fluorescence microscope. The scale bar is 20 µm. ( E ) The intracellular localization, ( F ) protein synthesis, ( G ) interaction, and ( H ) PLA of 3F-YPEL2 and/or HA-SQSTM1 in transiently transfected COS7 cells were as assessed as described in legend of A-D. HDAC1 was used as a loading control.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining, Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, DNA Amplification, Fluorescence, Microscopy

( A ) To examine whether or not a reduction of ELAVL1 levels in cells alters the location of YPEL in SGs, COS7 cells were left un-transfected (UT) or transiently transfected with a control siRNA AllStar (AS-siR) or a siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h. Cytoplasmic and nuclear extracts (50 µg) were subjected to SDS-10%PAGE for WB analysis using a monoclonal antibody specific to ELAVL1 (SCBT, sc-5261). HDAC1 probed with the HDAC1-specific antibody (Abcam, ab19845) was used as the loading control. ( B ) Transfected COS7 cells with the AllStar siRNA or the ELAVL1 siRNA pool for 24 h were also treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h. Cells were then fixed and subjected to ICC using the ELAVL1 antibody or the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( C & D ) To assess the co-localization of ELAVL1 and YPEL in SGs when ELAVL1 protein levels were reduced, COS7 cells transfected with ( C ) the control (AS-siR) or with ( D ) the siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h were treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h and subjected to ICC with the ELAVL1-specific monoclonal antibody and the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( E ) To assess the effect of the reduced levels of ELAVL1 on the size and numbers of SG, cells shown in Fig. D were subjected to quantification using the CellProfiler image analysis program. Asterisks indicate significant changes (P< 0.0001).

Figure Legend Snippet: ( A ) To examine whether or not a reduction of ELAVL1 levels in cells alters the location of YPEL in SGs, COS7 cells were left un-transfected (UT) or transiently transfected with a control siRNA AllStar (AS-siR) or a siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h. Cytoplasmic and nuclear extracts (50 µg) were subjected to SDS-10%PAGE for WB analysis using a monoclonal antibody specific to ELAVL1 (SCBT, sc-5261). HDAC1 probed with the HDAC1-specific antibody (Abcam, ab19845) was used as the loading control. ( B ) Transfected COS7 cells with the AllStar siRNA or the ELAVL1 siRNA pool for 24 h were also treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h. Cells were then fixed and subjected to ICC using the ELAVL1 antibody or the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( C & D ) To assess the co-localization of ELAVL1 and YPEL in SGs when ELAVL1 protein levels were reduced, COS7 cells transfected with ( C ) the control (AS-siR) or with ( D ) the siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h were treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h and subjected to ICC with the ELAVL1-specific monoclonal antibody and the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( E ) To assess the effect of the reduced levels of ELAVL1 on the size and numbers of SG, cells shown in Fig. D were subjected to quantification using the CellProfiler image analysis program. Asterisks indicate significant changes (P< 0.0001).

Techniques Used: Transfection, Control, Staining

Negative Control:

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation
Article Snippet: .. The antibodies used were as follows: rabbit anti-p63α (D2K8X; Cell Signaling), rabbit anti-HDAC1 (ab19845 Abcam), rabbit Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) (ab47915 Abcam), rabbit anti-SFPQ (Sigma-Aldrich PLA0181), rabbit anti-histone H3 (tri methyl K4) (ab8580 Abcam), rabbit IgG and mouse IgG (Invitrogen, 492024) (negative control). ..

Proximity Ligation Assay:

Article Title: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
Article Snippet: .. Primary antibodies used for PLA are: Anti-Biotin antibody (A150-109A, Bethyl Laboratories), Anti-Biotin antibody (AB_2339006, JacksonImmunoResearch), Anti-H3K9me3 (EPR16601) (Ab176916, Abcam), Anti-H3K9me2 (Ab1220, Abcam), Anti-H3K9me1 (EPR16989) (Ab176880, Abcam), Anti-G9a (EPR18894) (Ab 185050, Abcam), Anti-HDAC1 (Ab19845, Abcam), Anti-BRCA1 (D-9) (SC6954, Santa Cruz Biotechnology), Anti-BARD1 (A300-263A, Bethyl), Anti-RPA32/RAP2 (9H8) (Ab2175, Abcam), Anti-PCNA (PC10) (ab29, Abcam), Anti-H4K20me0 (EPR22116) (Ab227804, Abcam), Anti-H4K16ac (EPR1004) (Ab109463, Abcam), Anti-RAD51 (70-002, Bio Academia), Anti- H2AK15 ub (EDL H2AK15-4) (MABE1119, Millipore). ..

Activity Assay:

Article Title: Cathepsin L regulates oocyte meiosis and preimplantation embryo development
Article Snippet: .. Lactoferrin from bovine milk (LF; #L9507, Sigma) was dissolved in water and added to IVM/IVC medium at 10, 100 or 1000 μg/mL to inhibit CTSL activity in good quality oocytes/embryos while recombinant human active CTSL (rCTSL; #ab198444, Abcam, MA, USA) was supple- mented to IVM/IVC medium at 100, 200 or 400 ng/mL to enhance CTSL activity in good quality oocytes/embryos. .. Lactoferrin from bovine milk (LF; #L9507, Sigma) was dissolved in water and added to IVM/IVC medium at 10, 100 or 1000 μg/mL to inhibit CTSL activity in good quality oocytes/embryos while recombinant human active CTSL (rCTSL; #ab198444, Abcam, MA, USA) was supple- mented to IVM/IVC medium at 100, 200 or 400 ng/mL to enhance CTSL activity in good quality oocytes/embryos.

Recombinant:

Control:

Article Title: The growth hormone receptor interacts with transcriptional regulator HMGN1 upon GH-induced nuclear translocation
Article Snippet: .. Antibodies used in the experiments were GHR extracellular domain (Abcam, ab89400) GHR intracellular domain (Santa Cruz Biotechnology, sc-137185), HMGN1 (Invitrogen Life Technologies, 720,387), SUMO1 (Invitrogen Life Technologies, 332,400), anti-HDAC1 (Abcam, ab19845) phospho-STAT5 (pTyr694) (Life Technologies; 71–6900), STAT5 (C-17) antibody (Santa Cruz Biotechnology; sc-835), β-actin (Sigma-Aldrich;A1978); goat anti-mouse IgG (Sigma-Aldrich, A4416), goat anti-mouse Alexa Fluor 488 (Invitrogen, A-11001) and goat anti-rabbit IgG (Sigma-Aldrich, A3687) secondary antibodies; and isotype control antibody (ThermoFisher Scientific, 31,903). ..

Image Search Results

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) Based on data from the DepMap portal, the averag e expression of the YPEL gene family members relative to the expression of YPEL1 in breast cancer cell lines (DM-BCCL) or only in MCF7 cells (DM-MCF7) is shown. For comparison, our RT-qPCR analyses for the relative expression of YPEL genes in MCF7 cells (MCF7) are also indicated. ( B ) To assess whether or not E2-ERα signaling is involved in the regulation of YPEL2 , 3 , and 5 expressions, MCF7 cells grown for 72 h in CD-FBS containing growth medium were treated without (EtOH as control) or with 10 -9 M E2, and/or 10 -9 M ICI, a complete ER antagonist, for 24h. cDNAs generated from total RNA were subjected to qPCR. We also used the E2-ERα responsive gene TFF1 as a control. Star indicates a significant change compared to EtOH control. ( C ) Relative to YPEL1 , the expression of YPEL genes in COS7 cells was assessed by qPCR using cDNA from total RNA obtained from COS7 cells grown in steady-state conditions. ( D ) To evaluate the synthesis of YPEL1-5 proteins, which Y3.1 and Y3.2 indicate short and long variant YPEL3 proteins, COS7 cells were transiently transfected with the expression vector pcDNA3.1(-) bearing none (EV) or cDNA for a YPEL protein. Total protein extracts of COS7 cells grown in steady-state condition were subjected to SDS-15%PAGE followed by WB using a pan-YPEL antibody (SCBT, sc99727) and an HRP-conjugated goat-anti-rabbit secondary antibody (Advansta R-05072–500). Membranes were re-probed with an antibody specific to HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( E ) To assess the synthesis of endogenous YPEL proteins in COS7 cells, protein extracts ranging from 50 to 150 µg were subjected to WB using the pan-YPEL antibody. We also used protein extracts from COS7 cells transiently transfected with pcDNA3.1(-) bearing the 3F-YPEL2 cDNA for comparison. Membranes were re-probed with the HDAC1 antibody (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( F ) To assess the intracellular localization of endogenous YPEL (YPEL) proteins, COS7 cells grown on coverslips were subjected to ICC using the pan-YPEL antibody followed by an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Abcam, ab150080) or a vimentin (sc-6260) antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113). DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( G & H ) The intracellular localization of 3F-YPEL2 was evaluated in transiently transfected cells with the use of the Flag ( G ) or the pan-YPEL ( H ) antibody or GFP fusion ( I ). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Article Snippet: Rabbit polyclonal HDAC1 (ab19845), rabbit polyclonal β-actin (ab8227), rabbit polyclonal biotin (ab53494) antibodies, and Alexa Fluor 647-conjugated goat anti-rabbit IgG (ab150083) were acquired from Abcam Inc. (Connecticut, USA).

Techniques: Expressing, Comparison, Quantitative RT-PCR, Control, Generated, Variant Assay, Transfection, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) COS7 cells were transiently transfected with the pINDUCER20-MCS vector bearing none (EV) or the 3F-YPEL2 cDNA for 24 h. Cells were then treated without (0) or with varying concentrations (1-1000 ng/ml) of Dox for 24 h. Total cell extracts (50 µg) were then subjected to WB using the Flag antibody followed by an HRP-conjugated goat-anti-mouse secondary antibody (Advansta R-05071-500). Membranes were re-probed with the HDAC1 antibody. Molecular masses (MM) in kDa are indicated. ( B ) Transiently transfected cells with pINDUCER20-MCS bearing the 3F-YPEL2 cDNA for 24 h were then treated with 0, 5, 10, or 100 ng/ml Dox for 24 h. Cells were subsequently fixed, permeabilized, washed, and stained with the Flag antibody followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Abcam, ab150113) or a Lamin B1 antibody (Abcam, ab16048) followed by an Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( C ) To assess the subcellular levels of inducibly synthesized 3F-YPEL2 in transiently transfected cells treated without or with various concentrations of Dox for 24 h, fractionated nuclear and cytoplasmic protein extracts were subjected to WB using the Flag antibody. Membranes were re-probed with an antibody specific to β-actin (Abcam, ab8227) or HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. ( D ) To assess the effects of 3F-YPEL2 on cellular growth, COS7 cells transiently transfected with pINDUCER20-MCS bearing none (EV) or the 3F-YPEL2 cDNA for 24 h were treated without or with 10 ng/ml Dox for 24 h intervals up to 96 h. At every 24 h, cells were collected and counted with a hematocytometer. The asterisk indicates significant changes (P<0.05) in cellular growth depicted as fold changes. ( E ) Crystal violet staining for cellular growth and ( F ) WB analysis with the Flag antibody of COS7 cells inducibly synthesizing 3F-YPEL2 at 96 h are shown.

Techniques: Transfection, Plasmid Preparation, Staining, Synthesized

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) COS7 cells were transiently transfected with the pINDUCER20-MCS vector bearing none (EV) or the 3F-YPEL2 cDNA for 24 h. Cells were then treated without or with 10 ng/ml of Dox every 24 h for 96 h. Total cell extracts (50 µg) were then subjected to WB using the Flag antibody. Membranes were re-probed with the HDAC1 antibody. Molecular masses (MM) in kDa are indicated. ( B ) Transfected cells treated without or with 10 ng/ml of Dox for 24 h were also subjected to cell cycle analysis and ( C ) Annexin V staining. ( D ) Un-transfected cells were also treated with 10 or 100 µM apoptosis inducer Doxorubicin for 24h as control.

Techniques: Transfection, Plasmid Preparation, Cell Cycle Assay, Staining, Control

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) COS7 cells were transfected with pINDUCER20-MCS carrying none (EV), the 3F-YPEL2 (3F-Y2), 3F-YPEL2-Turbo-HA (3F-Y2-T-HA), or Turbo-HA (T-HA) cDNA. 24 hours after transfection, cells were treated without or with 10 ng/ml Dox to induce protein synthesis for 24 h in the absence or presence of biotin (50 μM) for the biotinylation of endogenous proteins. Cells were then collected and equal amounts (50 µg) of protein extracts were subjected to SDS-10%PAGE electrophoresis followed by WB analyses using the Flag, HA, or Biotin (Abcam, ab53494) antibody. Molecular masses are indicated in kDa. ( B ) To assess subcellular distributions of 3F-YPEL2 (3F-Y2), Turbo-HA (T-HA), or 3F-YPEL2-Turbo-HA (3F-Y2-T-HA) proteins following 10 ng/ml Dox induction for 24 h in transiently transfected cells, cytoplasmic, nuclear, or the total protein extracts (50 µg) were subjected to WB analyses wherein β-actin (β-Ac, ab8227) and HDAC1 (HD1) were used as the cytoplasmic and nuclear protein loading controls. Molecular masses are depicted in kDa. ( C ) Intracellular locations of the TurboID constructs in the transiently transfected cell grown on coverslips without (-) with Biotin (+) were assessed with the Flag antibody followed by the Alexa Fluor 488-conjugated secondary antibody (Abcam, ab150113), or HA and/or biotin antibody followed by the Alexa Fluor 594-conjugated secondary antibody (Abcam, ab150080). DAPI staining indicates the nucleus. The scale bar is 20 µm.

Techniques: Transfection, Electrophoresis, Construct, Staining

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A-C ) To assess the interactions of 3F-YPEL2 with putative interaction partners, the expression vector pcDNA3.1(-) bearing the 3F-YPEL2, ( A ) HA-ADSS, ( B ) HA-EEF1D, or ( C ) HA-G3BP1 cDNA were transiently transfected into COS7 cells for 24h. The synthesis of proteins was assessed by WB using the Flag (F-1804) or the HA (ab9119) antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody (ab19845). Cellular extracts (500 μg) of transiently co-transfected cells were subjected to Co-IP with the Flag, HA, or isotype-matched IgG. The precipitates were then subjected to SDS-10%PAGE followed by WB using the HA or Flag antibody. Molecular masses (MM) in kDa are indicated.

Techniques: Expressing, Plasmid Preparation, Transfection, Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) To assess the intracellular localization of 3F-YPEL2 and/or HA-ELAVL1, COS7 cells grown on coverslips un-transfected (UT) or transiently transfected for 36 h with the expression vector pcDNA3.1(-) bearing the 3F-YPEL2 or HA-ELAVL1 cDNA were stained with the Flag or the HA antibody. DAPI was used to indicate the nucleus. The scale bar is 20 µm. ( B ) To examine the protein synthesis, COS7 cells were transfected (+) with the expression vector bearing 3F-YPEL2 and/or HA-ELAVL1 cDNA. The synthesis of proteins was assessed by WB using the Flag or the HA antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody. ( C ). The cellular extracts (500 μg) of transiently co-transfected COS7 cells were subjected to Co-IP with the HA, Flag, or isotype-matched IgG. 50 μg of lysates was used as input control. The precipitates were subjected to SDS-10%PAGE followed by WB using the Flag or the HA antibody. Molecular masses (MM) in kDa are indicated. ( D ) To assess in cellula interaction of 3F-YPEL2 and HA-ELAVL1, the proximity ligation assay was carried out in transiently transfected COS7 cells grown on coverslips. Cells were fixed, permeabilized, blocked, and probed with the HA and/or the Flag antibody. Cells were then subjected to fluorescent probes for circular DNA amplification for proximity interaction foci. DAPI was used for nuclear staining. Images were captured with a fluorescence microscope. The scale bar is 20 µm. ( E ) The intracellular localization, ( F ) protein synthesis, ( G ) interaction, and ( H ) PLA of 3F-YPEL2 and/or HA-SQSTM1 in transiently transfected COS7 cells were as assessed as described in legend of A-D. HDAC1 was used as a loading control.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, DNA Amplification, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1101/2023.07.31.551286

Figure Lengend Snippet: ( A ) To examine whether or not a reduction of ELAVL1 levels in cells alters the location of YPEL in SGs, COS7 cells were left un-transfected (UT) or transiently transfected with a control siRNA AllStar (AS-siR) or a siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h. Cytoplasmic and nuclear extracts (50 µg) were subjected to SDS-10%PAGE for WB analysis using a monoclonal antibody specific to ELAVL1 (SCBT, sc-5261). HDAC1 probed with the HDAC1-specific antibody (Abcam, ab19845) was used as the loading control. ( B ) Transfected COS7 cells with the AllStar siRNA or the ELAVL1 siRNA pool for 24 h were also treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h. Cells were then fixed and subjected to ICC using the ELAVL1 antibody or the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( C & D ) To assess the co-localization of ELAVL1 and YPEL in SGs when ELAVL1 protein levels were reduced, COS7 cells transfected with ( C ) the control (AS-siR) or with ( D ) the siRNA pool specific to ELAVL1 transcripts (EL-siR) for 24 h were treated without (-SA) or with 200 µM sodium arsenite (+SA) for 1 h and subjected to ICC with the ELAVL1-specific monoclonal antibody and the pan-YPEL antibody. DAPI staining indicates the nucleus. The scale bar is 20 µm. ( E ) To assess the effect of the reduced levels of ELAVL1 on the size and numbers of SG, cells shown in Fig. D were subjected to quantification using the CellProfiler image analysis program. Asterisks indicate significant changes (P< 0.0001).

Techniques: Transfection, Control, Staining

a , Left: representative images of PLA depicting H3K9me3 presence at replication sites (H3K9me3-EdU PLA, red). Nuclei were counterstained with DAPI (blue). Right: distribution of the total intensity of all H3K9me3-EdU PLA spots per nucleus in wild-type cells (WT), G9a knockout cells (G9a−/−) and wild-type cells treated with 1 µM UNC0642 (UNC0642). Cells were labelled with EdU for 20 min and were either left untreated (UT), treated with 1 mM HU for 1 h (HU) or treated with 1 mM HU for 1 h and released from HU for 25 min and labelled with EdU for 20 min (Rel). ( n WT-UT = 2,436, n WT-HU = 2,212, n WT-REL = 2,340, n G9aKO-UT = 1,038, n G9aKO-HU = 1,168, n G9aKO-REL = 1,074, n UNC0642-UT = 2,413, n UNC0642-HU = 2,328, n UNC0642-REL = 2,315 cells analysed). b – d , Same as a but showing the distribution of PLA spot intensity per nucleus for H3K9me1-EdU PLA ( n WT-UT = 1,346, n WT-HU = 1,050, n WT-REL = 1,192, n G9aKO-UT = 1,543, n G9aKO-HU = 1,470, n G9aKO-REL = 1,630, n UNC0642-UT = 1,502, n UNC0642-HU = 1,296, n UNC0642-REL = 1,338 cells analysed) ( b ), H3K9me2-EdU PLA ( n WT-UT = 1,442, n WT-HU = 1,431, n WT-REL = 1,338, n G9aKO-UT = 1,321, n G9aKO-HU = 1,381, n G9aKO-REL = 1,380, n UNC0642-UT = 1,367, n UNC0642-HU = 1,490, n UNC0642-REL = 1,411 cells analysed) ( c ) and G9a-EdU PLA ( n WT-UT = 1,407, n WT-HU = 1,086, n WT-REL = 1,502, n G9aKO-UT = 1,510, n G9aKO-HU = 1,513, n G9aKO-REL = 1,510, n UNC0642-UT = 1,504, n UNC0642-HU = 1,505, n UNC0642-REL = 1,501 cells analysed) ( d ). e , Distribution of H3K9me3-EdU (left) or G9a-EdU (right) total PLA spot intensity per nucleus of wild-type cells treated (ATRi+) or not (ATRi−) with 10 µM ATR inhibitor and EdU labelled for 20 min followed by a 1 mM HU treatment for 1 h. For H3K9me3-EdU PLA: n HU− = 909, n HU+ = 931; for G9a-EdU PLA: n HU− = 869, n HU+ = 1,080 cells analysed. f , Same as a but showing the distribution of H3K9me3-EdU total PLA spot intensity per nucleus for the indicated conditions ( n ctl-UT = 1,509, n ctl-HU = 1,509, n ctl-REL = 1,506, n UNC0642-UT = 2,003, n UNC0642-HU = 1,529, n UNC0642-REL = 1,543, n siSUV39h1-UT = 1,514, n siSUV39h1-HU = 1,502, n siSUV39h1-REL = 1,500, n UNC0642+siSUV39h1-UT = 1,502, n UNC0642+siSUV39h1-HU = 1,523, n UNC0642+siSUV39h1-REL = 1,507, cells analysed) (note that, for a – f , blue dashed indicates mean of the distribution, **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, NS, non-significant, one-way analysis of variance Kruskal–Wallis test followed by Dunn’s test is used for all statistical analysis). g , Model summarizing G9a and SUV39h1 role at stalled replication forks. Upon replication stress, checkpoint-regulated G9a activity at stressed replication forks results in transient accumulation of H3K9me1/2 allowing SUV39h1 to catalyse H3K9me3 modification. Further accumulating HDAC1 resulted in the loss of H4K16ac. Figure created with biorender.com . h , Representative images of the changes over time of a stripe of photo-activated GFP-H2A for the indicated conditions. This experiment was reproduced independently three times with similar results. i , Mean photo-activated GFP-H2A area over time relative to the area at T = 0 min in percentage ± standard deviation. In PCNA negative (black) and positive (red) for untreated cell: WT-UT (left), cells undergoing replication stress: WT + HU (middle) and cells undergoing replication stress in the absence of G9a activity (right). Unpaired two-sided t -test, **** P ≤ 0.0001, ** P ≤ 0.01. For experimental design, see Extended Data Fig. . n = 3 independent experiments. Source numerical data are available in .

Journal: Nature Cell Biology

Article Title: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

doi: 10.1038/s41556-023-01167-z

Figure Lengend Snippet: a , Left: representative images of PLA depicting H3K9me3 presence at replication sites (H3K9me3-EdU PLA, red). Nuclei were counterstained with DAPI (blue). Right: distribution of the total intensity of all H3K9me3-EdU PLA spots per nucleus in wild-type cells (WT), G9a knockout cells (G9a−/−) and wild-type cells treated with 1 µM UNC0642 (UNC0642). Cells were labelled with EdU for 20 min and were either left untreated (UT), treated with 1 mM HU for 1 h (HU) or treated with 1 mM HU for 1 h and released from HU for 25 min and labelled with EdU for 20 min (Rel). ( n WT-UT = 2,436, n WT-HU = 2,212, n WT-REL = 2,340, n G9aKO-UT = 1,038, n G9aKO-HU = 1,168, n G9aKO-REL = 1,074, n UNC0642-UT = 2,413, n UNC0642-HU = 2,328, n UNC0642-REL = 2,315 cells analysed). b – d , Same as a but showing the distribution of PLA spot intensity per nucleus for H3K9me1-EdU PLA ( n WT-UT = 1,346, n WT-HU = 1,050, n WT-REL = 1,192, n G9aKO-UT = 1,543, n G9aKO-HU = 1,470, n G9aKO-REL = 1,630, n UNC0642-UT = 1,502, n UNC0642-HU = 1,296, n UNC0642-REL = 1,338 cells analysed) ( b ), H3K9me2-EdU PLA ( n WT-UT = 1,442, n WT-HU = 1,431, n WT-REL = 1,338, n G9aKO-UT = 1,321, n G9aKO-HU = 1,381, n G9aKO-REL = 1,380, n UNC0642-UT = 1,367, n UNC0642-HU = 1,490, n UNC0642-REL = 1,411 cells analysed) ( c ) and G9a-EdU PLA ( n WT-UT = 1,407, n WT-HU = 1,086, n WT-REL = 1,502, n G9aKO-UT = 1,510, n G9aKO-HU = 1,513, n G9aKO-REL = 1,510, n UNC0642-UT = 1,504, n UNC0642-HU = 1,505, n UNC0642-REL = 1,501 cells analysed) ( d ). e , Distribution of H3K9me3-EdU (left) or G9a-EdU (right) total PLA spot intensity per nucleus of wild-type cells treated (ATRi+) or not (ATRi−) with 10 µM ATR inhibitor and EdU labelled for 20 min followed by a 1 mM HU treatment for 1 h. For H3K9me3-EdU PLA: n HU− = 909, n HU+ = 931; for G9a-EdU PLA: n HU− = 869, n HU+ = 1,080 cells analysed. f , Same as a but showing the distribution of H3K9me3-EdU total PLA spot intensity per nucleus for the indicated conditions ( n ctl-UT = 1,509, n ctl-HU = 1,509, n ctl-REL = 1,506, n UNC0642-UT = 2,003, n UNC0642-HU = 1,529, n UNC0642-REL = 1,543, n siSUV39h1-UT = 1,514, n siSUV39h1-HU = 1,502, n siSUV39h1-REL = 1,500, n UNC0642+siSUV39h1-UT = 1,502, n UNC0642+siSUV39h1-HU = 1,523, n UNC0642+siSUV39h1-REL = 1,507, cells analysed) (note that, for a – f , blue dashed indicates mean of the distribution, **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, NS, non-significant, one-way analysis of variance Kruskal–Wallis test followed by Dunn’s test is used for all statistical analysis). g , Model summarizing G9a and SUV39h1 role at stalled replication forks. Upon replication stress, checkpoint-regulated G9a activity at stressed replication forks results in transient accumulation of H3K9me1/2 allowing SUV39h1 to catalyse H3K9me3 modification. Further accumulating HDAC1 resulted in the loss of H4K16ac. Figure created with biorender.com . h , Representative images of the changes over time of a stripe of photo-activated GFP-H2A for the indicated conditions. This experiment was reproduced independently three times with similar results. i , Mean photo-activated GFP-H2A area over time relative to the area at T = 0 min in percentage ± standard deviation. In PCNA negative (black) and positive (red) for untreated cell: WT-UT (left), cells undergoing replication stress: WT + HU (middle) and cells undergoing replication stress in the absence of G9a activity (right). Unpaired two-sided t -test, **** P ≤ 0.0001, ** P ≤ 0.01. For experimental design, see Extended Data Fig. . n = 3 independent experiments. Source numerical data are available in .

Article Snippet: Primary antibodies used for PLA are: Anti-Biotin antibody (A150-109A, Bethyl Laboratories), Anti-Biotin antibody (AB_2339006, JacksonImmunoResearch), Anti-H3K9me3 (EPR16601) (Ab176916, Abcam), Anti-H3K9me2 (Ab1220, Abcam), Anti-H3K9me1 (EPR16989) (Ab176880, Abcam), Anti-G9a (EPR18894) (Ab 185050, Abcam), Anti-HDAC1 (Ab19845, Abcam), Anti-BRCA1 (D-9) (SC6954, Santa Cruz Biotechnology), Anti-BARD1 (A300-263A, Bethyl), Anti-RPA32/RAP2 (9H8) (Ab2175, Abcam), Anti-PCNA (PC10) (ab29, Abcam), Anti-H4K20me0 (EPR22116) (Ab227804, Abcam), Anti-H4K16ac (EPR1004) (Ab109463, Abcam), Anti-RAD51 (70-002, Bio Academia), Anti- H2AK15 ub (EDL H2AK15-4) (MABE1119, Millipore).

Techniques: Knock-Out, Activity Assay, Modification, Standard Deviation

( a ) Plot showing distribution of H3K9me3-EdU total PLA spot intensity per nucleus for the indicated conditions. (n siCTL-UT = 925, n siCTL-HU = 951, n siCTL-REL = 990, n siSETDB1-UT = 1071, n siSETDB1-HU = 1040, n siSETDB1-REL = 1138 cells analyzed; blue dashed line represents the mean of the distribution, **** = P ≤ 0.0001, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( b ) Analysis of ChromStretch fibers to assess the dynamics of H3K9me1 at replication upon replication stress. Quantification of H3K9me1 signal overlapping with EdU for the indicated condition. The signal is represented as a fold increase compared to the mean H3K9me1 signal of the untreated condition. (n siCTL-UT = 100, n siCTL-HU = 100, n siSUV39h1-UT = 132, n siSUV39h1-HU = 100, n siSUV39h1+UNC0642-UT = 87, n siSUV39h1+UNC0642-HU = 100 cells analyzed; **** = P ≤ 0.0001, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( c ) Plot showing distribution of HDAC1-EdU total PLA spot intensity per nucleus for the indicated conditions. (n WT-UT = 1691, n WT-HU = 1871, n WT-REL = 1771, n UNC0642-UT = 1534, n UNC0642-HU = 1798, n UNC0642-REL = 1652 cells analyzed; blue dashed line represents the mean of the distribution, **** = P ≤ 0.0001, ** = P ≤ 0.01,Kruskal-Wallis test followed by Dunn’s test). ( d ) Plot showing distribution of H4K16ac-EdU total PLA spot intensity per nucleus for the indicated conditions. (n WT-UT = 1507, n WT-HU = 1254, n WT-REL = 1489, n UNC0642-UT = 1187, n UNC0642-HU = 1488, n UNC0642-REL = 1365 cells analyzed; **** = P ≤ 0.0001, ** = P ≤ 0.01, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( e ) Chromatin compaction can be followed in replicating (PCNA positive) and non-replicating (PCNA negative) cells in which a stripe of photo-activable GFP-H2A has been activated. Adding HU and/or UNC0642 immediately after the activation of GFP-H2A allow to measure over time the impact of these drugs on chromatin compaction. Figure created with biorender.com . Source numerical data are available in source data.

Journal: Nature Cell Biology

Article Title: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

doi: 10.1038/s41556-023-01167-z

Figure Lengend Snippet: ( a ) Plot showing distribution of H3K9me3-EdU total PLA spot intensity per nucleus for the indicated conditions. (n siCTL-UT = 925, n siCTL-HU = 951, n siCTL-REL = 990, n siSETDB1-UT = 1071, n siSETDB1-HU = 1040, n siSETDB1-REL = 1138 cells analyzed; blue dashed line represents the mean of the distribution, **** = P ≤ 0.0001, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( b ) Analysis of ChromStretch fibers to assess the dynamics of H3K9me1 at replication upon replication stress. Quantification of H3K9me1 signal overlapping with EdU for the indicated condition. The signal is represented as a fold increase compared to the mean H3K9me1 signal of the untreated condition. (n siCTL-UT = 100, n siCTL-HU = 100, n siSUV39h1-UT = 132, n siSUV39h1-HU = 100, n siSUV39h1+UNC0642-UT = 87, n siSUV39h1+UNC0642-HU = 100 cells analyzed; **** = P ≤ 0.0001, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( c ) Plot showing distribution of HDAC1-EdU total PLA spot intensity per nucleus for the indicated conditions. (n WT-UT = 1691, n WT-HU = 1871, n WT-REL = 1771, n UNC0642-UT = 1534, n UNC0642-HU = 1798, n UNC0642-REL = 1652 cells analyzed; blue dashed line represents the mean of the distribution, **** = P ≤ 0.0001, ** = P ≤ 0.01,Kruskal-Wallis test followed by Dunn’s test). ( d ) Plot showing distribution of H4K16ac-EdU total PLA spot intensity per nucleus for the indicated conditions. (n WT-UT = 1507, n WT-HU = 1254, n WT-REL = 1489, n UNC0642-UT = 1187, n UNC0642-HU = 1488, n UNC0642-REL = 1365 cells analyzed; **** = P ≤ 0.0001, ** = P ≤ 0.01, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). ( e ) Chromatin compaction can be followed in replicating (PCNA positive) and non-replicating (PCNA negative) cells in which a stripe of photo-activable GFP-H2A has been activated. Adding HU and/or UNC0642 immediately after the activation of GFP-H2A allow to measure over time the impact of these drugs on chromatin compaction. Figure created with biorender.com . Source numerical data are available in source data.

Techniques: Activation Assay

a , b , Combined mean expression was calculated to distinguish TCGA patients with ovarian cancer with low or high GLP/G9a expression , , . Kaplan–Meier curves were generated against progression-free survival ( a ) and overall patient survival ( b ) ( n = 614 patients). P values were calculated with the use of a two-sided log-rank test. c , G9a/EHMT2 associated with replication forks is activated by canonical DNA replication checkpoint pathway to catalyse H3K9me1/me2 at replication forks upon replication stress. Activated G9a generates a platform of H3K9me1/me2/me3 in concert with Suv39h1 at the site of stressed replication forks, which subsequently recruits histone deacetylase, HDAC1 to deacetylate the nucleosomes. Such closed chromatin conformation may create a protective compaction bubble that protects replication forks by (1) promoting efficient recruitment of fork protection factors, BARD1-BRCA1; and (2) such a conformation may also prevent the access to DNA nucleases and other detrimental factors, such as PRIMPOL that can lead to accumulation of ssDNA gaps behind the replication forks. Furthermore, synergistic activity of G9a and Suv39h1 further prevents the substrate, H3K9me1/me2 nucleosomes, availability to H3K9-demethylase, JMJD1A/KDM3A, timely assembly of which facilitates the disassembly of heterochromatin to promote their fork restart. Figure created with biorender.com . Source numerical data are available in .

Journal: Nature Cell Biology

Article Title: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

doi: 10.1038/s41556-023-01167-z

Figure Lengend Snippet: a , b , Combined mean expression was calculated to distinguish TCGA patients with ovarian cancer with low or high GLP/G9a expression , , . Kaplan–Meier curves were generated against progression-free survival ( a ) and overall patient survival ( b ) ( n = 614 patients). P values were calculated with the use of a two-sided log-rank test. c , G9a/EHMT2 associated with replication forks is activated by canonical DNA replication checkpoint pathway to catalyse H3K9me1/me2 at replication forks upon replication stress. Activated G9a generates a platform of H3K9me1/me2/me3 in concert with Suv39h1 at the site of stressed replication forks, which subsequently recruits histone deacetylase, HDAC1 to deacetylate the nucleosomes. Such closed chromatin conformation may create a protective compaction bubble that protects replication forks by (1) promoting efficient recruitment of fork protection factors, BARD1-BRCA1; and (2) such a conformation may also prevent the access to DNA nucleases and other detrimental factors, such as PRIMPOL that can lead to accumulation of ssDNA gaps behind the replication forks. Furthermore, synergistic activity of G9a and Suv39h1 further prevents the substrate, H3K9me1/me2 nucleosomes, availability to H3K9-demethylase, JMJD1A/KDM3A, timely assembly of which facilitates the disassembly of heterochromatin to promote their fork restart. Figure created with biorender.com . Source numerical data are available in .

Techniques: Expressing, Generated, Histone Deacetylase Assay, Activity Assay

A Schematic illustration of the lncRNAs array-based approach utilized to identify lncRNAs regulated by p63. Briefly, the indicated primary (HEKn and hMEC) and cancer cell types (A253, FaDu, HCC1954) were transfected with scramble (SCR) or siRNA oligos targeting p63 mRNA (sip63), and the cDNA utilized for hybridization assay of a custom-made lncRNAs array. B Venn diagrams showing shared downregulated and upregulated lncRNAs in sip63-transfected cells. C Human primary keratinocytes (HEKn) were transfected with siRNA targeting p63 (sip63), ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). MALAT1 and NEAT1 RNA levels were quantified by RT-qPCR (left panel). Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). D HEKn cells were transfected with siRNA targeting ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). NEAT1 long isoform (NEAT1_2) RNA levels were quantified by RT-qPCR. Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. E ChIP-seq enrichment of endogenous p63 at MALAT1 and NEAT1 genomic loci in HEKn cells (GSM1446927). F ChIP-qPCR showing ΔNp63 occupancy at the p63 binding site of MALAT1 and NEAT1 genomic loci. Average values from n = 2 biological replicates measured using three technical replicates are plotted. G ChIP-qPCR showing endogenous HDAC1 occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells. Average values from n = 2 biological replicates measured using three technical replicates are plotted. H ChIP-qPCR showing Histone H3 acetylated (H3ac) occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells transfected with scramble (SCR) or siRNA oligo targeting p63 (sip63) (left panels). Average values from n = 2 biological replicates measured using three technical replicates are plotted. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation

doi: 10.1038/s41467-023-39011-5

Figure Lengend Snippet: A Schematic illustration of the lncRNAs array-based approach utilized to identify lncRNAs regulated by p63. Briefly, the indicated primary (HEKn and hMEC) and cancer cell types (A253, FaDu, HCC1954) were transfected with scramble (SCR) or siRNA oligos targeting p63 mRNA (sip63), and the cDNA utilized for hybridization assay of a custom-made lncRNAs array. B Venn diagrams showing shared downregulated and upregulated lncRNAs in sip63-transfected cells. C Human primary keratinocytes (HEKn) were transfected with siRNA targeting p63 (sip63), ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). MALAT1 and NEAT1 RNA levels were quantified by RT-qPCR (left panel). Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). D HEKn cells were transfected with siRNA targeting ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). NEAT1 long isoform (NEAT1_2) RNA levels were quantified by RT-qPCR. Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. E ChIP-seq enrichment of endogenous p63 at MALAT1 and NEAT1 genomic loci in HEKn cells (GSM1446927). F ChIP-qPCR showing ΔNp63 occupancy at the p63 binding site of MALAT1 and NEAT1 genomic loci. Average values from n = 2 biological replicates measured using three technical replicates are plotted. G ChIP-qPCR showing endogenous HDAC1 occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells. Average values from n = 2 biological replicates measured using three technical replicates are plotted. H ChIP-qPCR showing Histone H3 acetylated (H3ac) occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells transfected with scramble (SCR) or siRNA oligo targeting p63 (sip63) (left panels). Average values from n = 2 biological replicates measured using three technical replicates are plotted. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). Source data are provided as a Source Data file.

Article Snippet: The antibodies used were as follows: rabbit anti-p63α (D2K8X; Cell Signaling), rabbit anti-HDAC1 (ab19845 Abcam), rabbit Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) (ab47915 Abcam), rabbit anti-SFPQ (Sigma-Aldrich PLA0181), rabbit anti-histone H3 (tri methyl K4) (ab8580 Abcam), rabbit IgG and mouse IgG (Invitrogen, 492024) (negative control).

Techniques: Transfection, Hybridization, Quantitative RT-PCR, Two Tailed Test, Western Blot, ChIP-sequencing, Binding Assay

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rabbit polyclonal hdac1 (Danaher Inc)

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